Nanoparticles in
biomolecular detection
The use of nanoparticles as labels in biomolecular detection in place of
conventional molecular fluorophores has led to improvements in
sensitivity, selectivity, and multiplexing capacity. Nevertheless, further
simplification is needed to take these technologies from the laboratory
to point of care. Here, we summarize the latest developments in the use
of nanoparticles as labels, especially in bioaffinity sensors for the
detection of nucleic acids and proteins.
Natalia C. Tansil and Zhiqiang Gao*
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138699
School of Materials Science and Engineering, Nanyang Technological University, Blk N4.1, Nanyang Avenue, Singapore 639798
*E-mail: [email protected]
Over the past decade, many important technological advances have
drawbacks of using molecular fluorophores apply. Because of the low
been made in the use of nanotechnology for biomolecular
abundance of protein and the limited number of procedures for protein
detection1,2. Coupled with the development of optical,
amplification, sensitivity needs to be improved.
electrochemical, and various other techniques for monitoring
Nanoparticles, in particular, have been used extensively in bioaffinity
biorecognition events on solid devices and in solution, a lot of effort
sensors for nucleic acids and proteins3,4. These particles are unique
has been put into realizing accurate, sensitive, selective, and practical
because their nanometer size gives rise to a high reactivity and
biosensing devices for both laboratory and point-of-care applications.
beneficial physical properties (electrical, electrochemical, optical, and
Currently, DNA assays rely on a combination of amplification by
magnetic) that are chemically tailorable. Their usage can potentially
polymerase chain reaction (PCR) and detection using molecular
translate into new assays that improve on current methods of DNA and
fluorophores as labels. The typical detection limit is in the picomolar
protein detection, as will be discussed in this article. Nanofabrication
range. Arrays containing thousands of unique probe sequences have also
and other nanostructures such as nanowires and nanotubes are beyond
been constructed. However, several major drawbacks still remain. Broad
the scope of this review.
absorption and emission bands and nonuniform rates of fluorophore
photobleaching may reduce accuracy. Sophisticated algorithms and
Au nanoparticles
expensive instrumentation are needed for fluorescence readout,
Optical detection
restricting application to laboratories. Furthermore, the selective and
In a milestone discovery in 1996, Mirkin et al.5 reported unique optical
nonlinear target amplification in PCR may distort gene expression.
and melting properties for an aggregate of Au nanoparticles and
Proteins are generally detected using immunoassays. Enzyme-linked
28
oligonucleotides. Specific interactions between oligonucleotides brought
immunosorbent assay (ELISA) remains the gold standard in protein
about the assembly of the 13 nm Au nanoparticles to which they were
detection, with detection limits in the picomolar range. Again, the
attached. A color change was observed, caused by the aggregate
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Nanoparticles in biomolecular detection
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scattering properties and the interaction between particle surface
plasmon-resonant particles, are quench resistant and generate very high
plasmons as the distance between Au nanoparticles varied. This
signal intensities (a 60 nm Au particle is equivalent to 3.3 x 105
distance-dependent optical property has seen the use of Au
fluorescein molecules). Attaching biomolecules, such as antibodies and
nanoparticles in a plethora of biomolecular detection methods, started
DNA, to these nanoparticles does not affect their optical properties.
off by colorimetric systems6,7.
The nanoparticles have thus been used successfully as labels in nucleic
In DNA detection, Au nanoparticles are modified with
acid13 and protein detection14. More recently, colorimetric detection
oligonucleotide detection probes and introduced into a solution of the
based on evanescent-wave-induced light scattering has been
single-stranded target oligonucleotide. A polymeric network of
developed15,16. The 50 nm Au nanoparticle labels on the detection
nanoparticles is formed and a color change can be detected visually.
probes scatter green light, but switch to red upon hybridization with
Spotting the test solution on a white support enhances the color
target nucleic acids. Combining this technique with waveguide
contrast and provides a permanent record of the test, with a detection
detection, 333 fM of oligonucleotides and 33 fM of genomic DNA have
limit of 10 nM. Subsequent reports have confirmed that the
been detected15.
nanoparticle-oligonucleotide aggregates also exhibit an unusually sharp
The combination of Au nanoparticles and Raman spectroscopy is
melting transition6,8,9. Complementary target sequences can thus be
attractive because of: (1) the greatly amplified Raman scattering upon
better distinguished from one-base-mismatched sequences through a
adsorption of Raman dyes on metallic nanoparticles (surface-enhanced
thermal stringency wash. The level of selectivity allows one-pot
Raman scattering, or SERS); and (2) the narrow spectral characteristics
colorimetric detection of a target sequence in a complex mixture7.
of Raman dyes, which allow multiplexed detection. Raman dyes and
The sensitivity of colorimetric detection is improved by catalytic
detection probes are first attached to Au nanoparticles and hybridized
deposition of Ag on Au nanoparticle labels. Scanometric array detection,
with the captured targets. An Ag coating is then catalytically deposited
illustrated in Fig. 1, has been achieved using a flatbed scanner to detect
on the Au nanoparticles to promote SERS of the Raman dyes, and the
the Ag-enhanced labels with a detection limit of 50 fM10, 100 times
amplified signal is captured by spectroscopy. To prevent particle
more sensitive than the fluorophore-based system.
agglomeration and protect the Raman dyes, Mulvaney et al.17 deposited
An extension of scanometric detection – bio-barcode amplification –
a glass shell on the nanoparticles. This method has been used in the
was reported recently11. This involves magnetic microparticles with
multiplexed detection of nucleic acids18,19 and proteins17, with an
capture probes and Au nanoparticles with both reporter probes and
unoptimized detection limit of 20 fM for nucleic acids18. Compared with
numerous barcode oligonucleotides. The presence of target molecules
colorimetric and scanometric detection, this method offers more
that match both capture- and reporter-probes results in the formation
multiplexing capabilities afforded by the narrow-band spectroscopic
of a magnetic microparticle-Au nanoparticle sandwich that is
fingerprint of the Raman dyes. The absence of photobleaching is an
subsequently isolated from the system. The barcode oligonucleotides
advantage compared with fluorophore labeling.
are then dissolved and detected using scanometric detection with
In 1997, Kubitschko et al.20 proposed the use of 85 nm latex
Ag amplification, from which a detection limit as low as 500 zM has
nanoparticles as mass labels to enhance the refractometric signal in an
been obtained. This method is applicable to both DNA and proteins.
immunoassay. Building on a similar principle, surface plasmon resonance
Au and Ag nanoparticles also have very high light-scattering power,
as first reported by Yguerabide et al.12. These particles, also called
(a)
(SPR) measurement was recently adopted alongside Au nanoparticle
labeling. The nanoparticles bring about an amplified shift in angle because
(b)
(c)
Fig. 1 Scanometric DNA assay10. (a) Immobilization of capture probes on electrode. (b) Hybridization with target DNA and labeled detection probe.
(c) Amplification by reductive deposition of Ag followed by scanometric detection. (Reprinted with permission from10. © 2000 AAAS.)
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Nanoparticles in biomolecular detection
(a)
(b)
(c)
Fig. 2 Optical detection with Au nanoparticles as fluorescent quenchers27. (a, b) Owing to the conformation, the fluorescence labels are in close proximity to the
Au nanoparticles and their signals are quenched. (c) Upon hybridization, the rigid double-helical DNA molecules ‘open up’ their conformation and fluorescence is
restored. (Reprinted with permission from27. © 2004 American Chemical Society.)
they: (1) have a high dielectric constant; (2) cause electromagnetic
Fluorophore-tagged oligonucleotides are attached to the nanoparticles,
coupling with the SPR Au film; and (3) increase the surface mass. This
forming arch-like conformations that cause quenching of fluorophores
results in more sensitive detection. SPR biosensing using Au nanoparticle
on the particle surface. The binding of target molecules results in a
enhancement has been reported for the detection of nucleic acids21 as
conformational change that restores the fluorescence signal, as
well as proteins22-25. Detection limits for 24 mer oligonucleotides21 and
depicted in Fig. 2.
human immunoglobulin G22 were in the picomolar range. Earlier this
year, Aslan et al.26 reported a real-time bioaffinity monitoring system
Electrical detection
based on an angular-ratiometric approach to plasmon-resonance light
Direct electrical detection is one of the simplest methods for
scattering. As nanoparticles grow through bioaffinity reactions, they
bioaffinity sensing28. In this scheme (Fig. 3), capture probes are
deviate from Rayleigh theory and scatter more light in a forward
immobilized in micron-sized gaps between electrodes in a DNA array.
direction relative to the incident geometry. By comparing the scattered
Hybridization with target DNA and Au nanoparticle-labeled detection
light intensity at 90° and 140° relative to the incident light, the authors
probes localizes the nanoparticles in the gap, while subsequent Ag
managed to monitor the formation of aggregates of biotinylated 20 nm
deposition creates a ‘bridge’ across the gap. The detection of a
Au nanoparticles as they are crosslinked by the addition of streptavidin.
conductivity change results in a detection limit of 500 fM. A mutation
Au nanoparticles can also function both as a scaffold and
fluorescence quencher for the homogenous detection of nucleic acids27.
(a)
selectivity ratio of ~100 000:1 is also achieved by exploiting the
unique salt-concentration-dependent hybridization of the labeled
(b)
(c)
Fig. 3 Electrical detection of DNA hybridization using Au nanoparticle labels28. (a) Immobilization of capture probes in the gap between two electrodes.
(b) Hybridization with target DNA and Au nanoparticle-labeled detection probe. (c) Reductive deposition of Ag, creating a bridge that decreases resistance.
(Reprinted with permission from28. © 2002 AAAS.)
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probes. This is much higher than the selectivity level in the
nucleotides labeled with Au nanoparticles. If the SNP contains a certain
fluorescence-based approach (2.6:1) and the previously described
base that matches the labeled monobase, Au nanoparticles accumulate
scanometric approach (11:1). The feasibility of detecting single
on the electrode surface in the presence of DNA polymerase I. This is
nucleotide polymorphisms (SNPs) using this method has been confirmed
indicated by a significant increase in the Au oxidation wave. A model
by Burmeister et al.29. The same group has devised a method for
study was performed successfully on a synthetic 21 base
continuously monitoring the autometallographic enhancement
oligonucleotide target (Fig. 5).
process, eliminating the need for multistep enhancement and all the
washing, drying, and measurement cycles in between30.
Another signal amplification strategy is to attach electroactive
6-ferrocenylhexanethiol molecules onto the Au nanoparticle labels36,37.
A further improvement in the electrical detection method has been
Hybridization brings these compounds into close proximity with the
reported for immunoassays. By reducing the size of the electrode gap to
underlying electrode, causing a reversible electron-transfer reaction.
below 100 nm, a conductivity change can be detected even without Ag
With detection limits in the picomolar range, this method is simpler
enhancement31.
than the Ag-enhancement scheme, but just as sensitive33. As a side
note, remarkable sensitivity was achieved when a larger number of
Electrochemical detection
ferrocene units were packed into a micron-sized polystyrene bead38.
The redox properties of Au nanoparticles have led to their widespread
Electrochemical stripping is a sensitive electrochemical measurement
use as electrochemical labels in nucleic acid detection, with numerous
protocol. It was first used alongside Au nanoparticle labels in 200139,40.
configurations being explored. Ozsos et al.32 immobilized target DNA
Hybridization of target oligonucleotides to capture probes is followed by
on an electrode, followed by hybridization with complementary probes
binding of the Au nanoparticle labels, dissolution of the labels, and the
labeled with Au nanoparticles. The hybrid displayed a Au oxide wave at
potentiometric stripping measurement of the dissolved metal ions on an
around 1.20 V (Fig. 4).
electrode. Initially, a PCR step was needed, and the detection limit for
an amplified 406 base pair human cytomegalovirus DNA fragment was
Initially, this method was not sensitive enough, such that PCR
amplification was still needed. The detection limit for the PCR
5 pM of PCR product41. Precipitation of Au or Ag onto the Au
amplicons of Factor V Leiden mutation was 0.78 fM. Signal
nanoparticle labels leads to PCR-free detection with amplified signals
amplification can again be achieved by Ag enhancement33. Au-catalyzed
and lower detection limits41,42. In protein detection, Schistosoma
deposition of Ag on the nanoparticle labels brings a large number of Ag
japonicum antibody43, human immunoglobulin G44, and rabbit
immunoglobulin G45 have been detected. An optimized detection limit
of 0.25 pg/ml (1.5 pM) and a dynamic range of 1-500 pg/ml have been
reported45. This is 100 times more sensitive than conventional
immunoassays.
A common problem in Ag enhancement is the high background
signal as a result of nonspecific precipitation of Ag onto the substrate
electrode. This can be avoided by coating the electrode with multilayer
films of polyelectrolyte or by using materials with low Ag-enhancing
properties, such as indium tin oxide46. Various electrode surface
atoms closer to the electrode. A detection limit of 50 pM and selectivity
against one-base mismatches were achieved.
In another configuration, Au nanoparticle labels are attached to a
single-stranded-DNA binding protein34. Hybridization of capture probes
with matching targets hinders the binding of labeled proteins, as
indicated by the decrease in the Au redox signal. A detection limit of
2.17 pM was achieved.
Kerman et al.35 reported a strategy to not only detect the presence
of an SNP but also identify the bases involved using monobase
(a)
(b)
(c)
Fig. 4 Electrochemical detection of DNA hybridization using Au nanoparticle labels32. (a) Immobilization of target DNA. (b) Hybridization with Au nanoparticlelabeled detection probe. (c) Voltammetric detection of Au redox signal.
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Nanoparticles in biomolecular detection
magnetic beads for the control of bioelectrocatalytic processes in DNA
and protein detection. The beads in these cases do not function as
labels, and thus are outside the scope of this review.
Aside from Au, Ag53,54 and Cu/Au alloy55 nanoparticles have also
been used as oligonucleotide labeling tags in conjunction with stripping
electrochemical detection.
Other detection methods
In addition to optical and electrochemical detection, Au nanoparticle
labels have been used in combination with other detection platforms.
Scanning electrochemical microscopy56, based on the increase in
conductivity in the assay spot, is fairly sensitive. However, its
application may be limited by low sample throughput (a scan of an
area of 0.24 cm x 0.24 cm lasts 38 minutes). When used together with
a quartz crystal microbalance (QCM), Au nanoparticle labels result in a
detection limit of 1 fM57. This is more sensitive than any other QCMbased method available to date58. In an immunoassay, Au nanoparticle
labels were detected through a chemiluminescent reaction59.
Compared with stripping voltammetry, this method may be more
sensitive because of the higher sensitivity of chemiluminescent
detection.
Quantum dots
Optical detection
Quantum dots (QDs) were initially used as fluorescent biological
labels60,61. These semiconductor nanoparticles have narrow, sizetunable, symmetric emission spectra and are photochemically stable.
These features, in addition to their binding compatibility with DNA and
proteins60,61, render QDs prime candidates to replace fluorophores as
Fig. 5 Electrochemical identification of a single-nucleotide polymorphism35.
biological labeling agents62. In fluorescence-based applications,
Four Au nanoparticle-labeled monobases are added in separate assays. The
comparison of the Au redox signal generated from each assay indicates the
identity of the mismatch. (Reprinted with permission from35. © 2004 American
Chemical Society.)
core-shell structured QDs are more favorable63-65. Many core-shell
QDs have been prepared by capping an emissive semiconductor core
(CdSe, CdTe, etc.) with a thin shell of a higher band gap material (ZnS,
CdS, ZnSe, etc.). This increases the photostability of the core and
treatments47,
and
electrochemically48
and enzymatically
controlled49
deposition methods of Ag have been reported, all of which aim to
reduce the Ag-related background signal and thus to increase the
sensitivity.
quantum yield66,67.
In nucleic acid assays, QDs take the role of the fluorophore labels.
SNP detection of TP53 DNA and multiallele detection for hepatitis B
Alternatively, signal amplification has been achieved by loading
numerous Au nanoparticles on a polymeric
bead50.
The nanoparticles
and hepatitis C viruses have been achieved in a microarray format68.
Detection was performed at room temperature within minutes, with
can later be catalytically enlarged, dissolved, and measured by
true-to-false signal factors greater than ten. Unique in situ detection of
electrochemical stripping. The combined amplification effects allow the
chromosome abnormalities or mutations has also been realized using
detection of DNA targets down to 300 amol. A further reduction in
QDs as optical labels69. In immunoassays, cancer-cell-marker protein
background signal, and hence improvement in sensitivity, has been
her270, diphtheria toxin, and tetanus toxin71 have been detected
achieved using magnetic beads for the immobilization of DNA capture
successfully using QD-labeled antibodies.
probes39 or capture antibodies51 (Fig. 6). Upon hybridization, the
32
prevents surface quenching of the excitons, hence increasing the
Hybrid inorganic-bioreceptor conjugates made of QD-protein
magnetic beads can be magnetically collected for easy discrimination
assemblies can also function as chemical sensors. In these assemblies,
against unhybridized DNA or nonbinding antibodies. It is worth noting
multiple copies of dye-labeled proteins self-assemble to each QD,
that Willner and coworkers52 have extensively studied the use of
enabling fluorescence resonance energy transfer (FRET) between the
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Nanoparticles in biomolecular detection
(a)
(b)
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(d)
(c)
(f)
(e)
Fig. 6 Electrochemical stripping detection of antigen using magnetic beads and Au nanoparticle labels51. (a) Immobilization of capture antibody on a magnetic bead.
(b) Binding with target antigen. (c) Recognition by detection antibody. (d) Nanoparticle-catalyzed enlargement by Ag deposition. (e) Collection of magnetic beads
and the antibody-Ag complex using an external magnet. (f) Stripping voltammetric detection of the collected Au/Ag nanoparticle label.
QD and the dye molecules. In a scheme proposed by Willard et al.72,
detection using a commercial scanner and two sets of nanocrystals with
biotinylated bovine serum albumin is attached to CdSe-ZnS
orthogonal emission77.
nanoparticles, followed by specific binding of tetramethylrhodamine-
In another set of experiments, Han et al.78 and Rosenthal79 have
labeled streptavidin. This binding event leads to an enhanced
used QDs as identification tags to recognize various DNA sequences in
tetramethylrhodamine fluorescence signal caused by FRET. In another
a wavelength-and-intensity multiplexed detection scheme. Differently
scheme, where a dark quencher dye is used at the protein binding site,
sized (and thus differently colored) ZnS-capped CdSe nanoparticles
the addition of analyte displaces the dye and restores the QD emission
were embedded into polymeric microbeads at precisely controlled
signal73. With two cyanine dyes, a two-step FRET takes place and the
ratios. Each of these unique microbeads codes for one particular
presence of analyte is signaled by the wavelength of emission73. These
capture probe sequence, while the same fluorophore was used to label
schemes are possible because of the size-tunable emission of QDs, such
the entire target oligonucleotide. Simultaneous reading of coding and
that they resonate with the absorption of the acceptor dyes. For this
target signal reveals the identity of targets that are present in the
application, a combination of spectroscopic and crystallographic
mixture, as illustrated in Fig. 7. The combination of QDs with ten
analyses has been developed to study the orientation of proteins on
intensity levels and six colors could theoretically code one million
QD
surfaces74.
DNA hybridization detection using QD labels has recently been
unique nucleic acid sequences or proteins, enabling massively parallel
and high-throughput analysis. However, the implementation depends
achieved using a novel technique combining surface plasmon field
on the uniformity and reproducibility of QD synthesis. It is also
enhancement and fluorescence spectroscopy75. In this technique,
important to note that in this protocol, in contrast to the previous
capture probes are immobilized on a metal substrate. Upon binding, the
protocols, QDs are used for identification rather than signal
surface plasmon resonance of the metal surface excites and enhances
generation.
the fluorescence of the QD label on the target DNA, resulting in
enhanced sensitivity. By applying this microscopic method in an array
Electrochemical detection
format, multiplexed hybridization detection has been achieved. This is
The use of QDs for electrochemical monitoring of DNA hybridization
facilitated by the broad absorption and sharp emission spectra of QDs,
was first reported by Wang et al.80 using CdS nanoparticles. Based on
allowing the excitation of multicolored QDs with a single light source.
an electrochemical stripping protocol analogous to those used with Au
Gerion et al.76 have demonstrated the feasibility of sorting four
nanoparticle labels, this method exploits the attractive stripping
different QDs into different locations on the basis of complementary
behavior of Cd or Pb ions. The nanoparticle-promoted Cd precipitation
DNA (cDNA) interaction. The same group later reported the application
enlarges the nanoparticle labels and amplifies the stripping signal,
of DNA-QD conjugates in a cDNA microarray for SNP and multiallele
resulting in a detection limit of 100 fM for a 32 base target DNA. This
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Fig. 7 Wavelength-and-intensity multiplexed detection of antibodies using QD-loaded beads78. The unique probe identification (ID) signal is used to identify
individual sequences, while the analyte signal is generated by an attached fluorophore. (Reprinted with permission from78. © 2001 Nature Publishing Group.)
is slightly more sensitive than the corresponding Au nanoparticle-based
multiple DNA targets84. By attaching PbS, CdS, and ZnS to various
system proposed by the same group39,42.
detection probe sequences and subsequently stripping the labels at
QD labels made of
PbS81
and
ZnS82
have also proven useful in
electrochemical stripping detection of DNA, with detection limits in the
various potentials, the different target sequences can be detected and
quantified (Fig. 8). The same strategy has also been applied in a
subpicomolar range. An amplification strategy involving the use of CdS
multiplexed immunoassay of proteins85, with measurements of four
nanoparticle-loaded carbon nanotubes as labels was reported
antigens achieved in a single run. A greater degree of multiplexing may be
subsequently83.
achieved in connection with the encapsulation of various proportions of
Further exploration of QDs with dissimilar oxidation potentials has led
to their use as ‘electrochemical codes’ in the simultaneous detection of
(a)
(b)
different nanoparticles into polymeric carrier beads – an electrochemical
equivalent of the optical coding proposed by Han et al.78.
(c)
(d)
Fig. 8 Multitarget electrochemical detection of DNA using QD labels84. (a) Introduction of probe-modified magnetic beads. (b) Hybridization with DNA targets.
(c) Second hybridization with QD-labeled probes. (d) Dissolution of QDs and electrochemical detection. (Reprinted with permission from84. © 2003 American
Chemical Society.)
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An exciting development has been reported recently involving the
REVIEW FEATURE
labels. These sensors focus on determining the areal density of the
use of QD labels for SNP identification86. Four monobases were
beads rather than counting individual beads97,98. In more recent
uniquely labeled with four different QDs and then captured in
research, nanosized labels have been used. For example, 200 nm
different combinations for specific SNPs, yielding a distinct electronic
magnetic labels were used recently in single-bead detection on a
fingerprint. As a result, in contrast with other electrochemical
submicron GMR sensor99. Single-bead detection of nanosized magnetic
methods35,87,88, SNP identification has been achieved in a single
labels has also been performed on spin-valve sensors100-103, with the
voltammetric run. This new protocol may eventually facilitate simple,
successful detection of 11 nm Co103 and 16 nm Fe3O4104 nanoparticles
fast, and cost-effective screening of important SNPs in high-throughput
being reported by Grancharov et al.105. Another development in
automated operations.
magnetoresistive sensors was made in 2005 through the use of an
QD labels have also been used in electrochemical impedance
spectroscopic (EIS)
detection89.
Detection is based on the change in
alternating current field to accelerate DNA hybridization106. Surfacebound DNA probes rapidly hybridize with magnetically labeled
electron transfer resistance between the electrode and a redox marker
complementary target DNA when oscillating external magnetic fields
in the solution. As expected, resistance increases upon hybridization.
are applied through current-carrying lines. In 2005, 12 nm manganese
This increase is amplified up to 100 times when the target DNA is
ferrite nanoparticles were synthesized and biofunctionalized for use in a
labeled with CdS nanoparticles, owing to the labels’ space resistance
magnetic tunnel junction based sensor105.
and semiconducting properties. The detection of resistance change by
EIS is thus made possible.
Magnetic nanoparticles
Silica nanoparticles
Optical detection
More attention has been focused on dye-doped silica nanoparticles
Magnetic detection
since their first use by Tan and coworkers107 as labels in DNA
Because of their extensive use for separation and preconcentration in
detection. The nanoparticles, synthesized by reverse microemulsion,
electrochemical and optical biosensors, magnetic beads are readily
comprise numerous organic dye molecules in a silica matrix. The result
available. In recent years, magnetic nanoparticles have been finding use
is better resistance against photobleaching and an amplified signal for
not only as carriers but also as labels that indicate binding events90.
each bound label. Unlike organic fluorophores, these nanoparticles
Superparamagnetic nanoparticles make ideal labels because they are
do not suffer from blinking. An impressive detection limit of 0.8 fM
readily magnetized to large magnetic moments, which facilitates
and a selectivity ratio of 14:1 against one-base mismatches have been
detection, and yet the mutual magnetic attraction can easily be
achieved.
switched off to prevent irreversible aggregation.
Chemla et al.91 have reported an immunoassay using magnetic
Unique core-shell-structured silica nanoparticles have been reported
by Zhou et al.108, in which the fluorophore molecules in the core are
nanoparticle labels. Target antigens are immobilized on a Mylar-film-
protected and separated from the conjugated biomolecules. These
coated well, to which a suspension of labeled antibody is added. Upon
nanoparticles are stable in both aqueous electrolytes and organic
applying a magnetic field in one-second pulses, the nanoparticles
solvents, hence they do not aggregate. When used as labels in DNA
develop a net magnetization that relaxes when the field is turned off.
detection, a detection limit of 1 pM and a dynamic range of about four
Detection is achieved by differentiating the relaxation process: unbound
orders of magnitude have been achieved. While this system is better
nanoparticles relax rapidly by Brownian rotation, while the bound
than molecular fluorophore-based systems, it is not as sensitive as
nanoparticles undergo a slow Néel relaxation, giving out a magnetic flux
optical detection using Au nanoparticles. Successful
that can be detected by a superconducting quantum interference device.
immunofluorometric assays109-111 and cell quantitation on the basis of
The ability to distinguish between bound and unbound labels obviates
antibody recognition112-114 have also been attempted using silica
the need for separation and allows homogeneous detection. The same
particles doped with fluorescein isothiocyanate110,111,114 and
method has also been used to detect bacteria in a suspension on the
tris(2,2’-bipyridyl)-ruthenium(II)109,112,113. The detection limits were
basis of antibody interaction92.
typically less than 1 ng/ml110,111.
Magnetoelectronics has recently emerged as a promising platform
technology for biosensors93,94. This technique is based on the detection
Electrochemical detection
of the magnetic fringe field given off by a magnetically labeled target
Doping the nanosized silica matrix with a large quantity of redox active
interacting with a complementary biomolecule bound to a magnetic
compounds such as tris(2,2-bipyridyl)cobalt(III)115 results in sensitive
field sensor. Efforts have focused primarily on the fabrication of various
electrochemical labels. A detection limit of 200 pM has been achieved.
sensor designs rather than on the nanoparticle labels. In earlier
This is a reasonable value considering the simplicity of this assay
attempts to detect DNA using sensors based on giant
compared with the more sensitive but multistep method using
magnetoresistance (GMR)95,96, micron-sized Fe2O3 beads were used as
Au-loaded polymeric beads50.
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Other nanoparticles
Silica nanoparticles, despite showing great promise in biomolecular
Aside from the widely used metal and semiconductor nanoparticles,
detection, need to be explored further in order to realize their full
several other materials have also been explored as labels in
potential as labels. The eventual use of these materials will depend on
biomolecular detection.
the ease with which they can be synthesized and manipulated.
Eu2O3 is a simple inorganic phosphor with a spectrally narrow red
In comparing the detection techniques, colorimetric and direct
emission and a long fluorescence lifetime. Harma et al.116 trapped
electrical approaches are among the simplest systems available.
chelated Eu in polystyrene nanoparticles, conjugated biomolecules on
However, there is still room for improvement in the sensitivity of these
their surfaces, and used them as labels for time-resolved bioassays.
methods. Optical detection methods suffer from the drawback of bulky
Subsequently, the detection of various biomolecules such as prostate-
and expensive instrumentation. Electrochemical methods, on the other
specific antigen117, viruses118, thyroid-stimulating hormone119, and
hand, have become more attractive owing to their simplicity, low cost,
DNA120
and excellent portability. Unfortunately, the current amplification
have all been demonstrated using the Eu-doped nanoparticles.
The detection limits are ~1 pg/ml for proteins117 and 6.1 x 104 copies
strategy for electrochemical signal generation often involves multiple
for DNA120. Simpler synthesis methods have also been proposed121,122.
steps of deposition and stripping. Hence, there is a tradeoff between
A novel lanthanide-based nanoparticle has been proposed123. These
nanoparticles consist of a
Tb3+-doped
Gd2O3 core (providing high
fluorescence intensity) in a functionalized polysiloxane shell (enabling
dispersion in aqueous solution and conjugation with biomolecules).
Organic fluorescent nanoparticles, formed by the precipitation of
simplicity and sensitivity. Within the scope of this review, it is not
possible to explore in detail the mechanisms and implications of each
detection system. For further reading, please refer to other articles125,126.
Tremendous opportunities exist in the application of nanoparticles
for biomolecular detection. Novel nanoparticles have been fabricated
1-pyrenebutyric acid, have also been used in fluorescence-based
that might be useful for molecular detection. For example, magnetic-QD
assays124. These are photochemically stable and water soluble, and
hybrid nanoparticles127 may offer a unique combination of sample
exhibit ideal excitation and emission spectra, high room-temperature
manipulation and sensitive detection with negligible interference. OsO2
fluorescence quantum yields, and long fluorescence lifetimes. Since
nanoparticles, which have electrocatalytic properties, have been
they do not require coating, synthesis is greatly simplified. Under
exploited for the chemically amplified detection of microRNA
optimized conditions, human serum albumin, bovine serum albumin,
(miRNA)128. The association of these nanoparticles with hybridized
and γ-immunoglobulin G (γ-IgG) can be detected with detection limits
miRNA molecules leads to the formation of an electrocatalytic system
in the nanogram per milliliter range by using such labels.
that generates a measurable current upon the addition of substrate to
the solution. Novel types of nanoparticle label could be developed by
Concluding remarks and future outlook
entrapping or immobilizing multiple bioluminescent enzymes, such as
Many recent articles have reported the potential of nanoparticles as
luciferase, on nanoparticle carriers. The addition of a substrate will result
labels in biomolecular detection. Compared with current state-of-the-
in a luminescent signal with little interference from the background.
art DNA detection using fluorophore labels and protein detection using
It is also interesting to compare the performance of nanoparticle
ELISA, significant advances have been made in terms of sensitivity,
labels to other labels in biomolecular detection, such as electrochemical
selectivity, and multiplexing capacity.
tags129 and dendrimers130. Nonetheless, the eventual acceptance of
Of the various materials, Au nanoparticles seem to be the most
nanoparticle-based detection schemes for diagnostic applications will
versatile and extensively studied. QDs can be detected using optical and
depend on how these novel schemes compare with the current gold
electrochemical detection platforms similar to those used for Au
standards, i.e. PCR and ELISA, in terms of simplicity, sensitivity,
nanoparticles, and make excellent substitutes for organic fluorophores.
specificity, and reliability.
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